Thyroid hormones, L-thyroxine (T4) and L-triiodothyronine (T3), regulate many different physiological processes in different tissues in vertebrates. Most of the actions of thyroid hormones are mediated by the thyroid hormone receptor (“TR”), which is a member of the nuclear receptor superfamily of ligand-activated transcription regulators. This superfamily also includes receptors for steroid hormones, retinoids, and 1,25-dihydroxy vitamin D3. These receptors are transcription factors that can regulate expression of specific genes in various tissues and are targets for widely used drugs, such as tamoxifen, an estrogen receptor partial antagonist. There are two different genes that encode two different TRs, TRα and TRβ. These two TRs are often co-expressed at different levels in different tissues. Most thyroid hormones do not discriminate between the two TRs and bind both with similar affinities.
Gene knockout studies in mice indicate that TRβ plays a role in the development of the auditory system and in the negative feedback of thyroid stimulating hormone by T3 in the pituitary, whereas TRα, modulates the effect of thyroid hormone on calorigenesis and on the cardiovascular system. The identification of TR antagonists could play an important role in the future treatment of hypothyroidism. Such molecules would act rapidly by directly antagonizing the effect of thyroid hormone at the receptor level, a significant improvement for individuals with hypothyroidism who require surgery, have cardiac disease, or are at risk for life-threatening thyrotoxic storm.
Thus, there remains a need for the development of compounds that selectively modulate thyroid hormone action by functioning as isoform-selective agonists or antagonists of the thyroid hormone receptors (TRs) would prove useful for medical therapy. Recent efforts have focused on the design and synthesis of thyroid hormone (T3/T4) antagonists as potential therapeutic agents and chemical probes. There is also a need for the development of thyromimetic compounds that are more accessible than the natural hormone and have potentially useful receptor binding and activation properties.
Thyroid hormone receptor preferentially binds 3,5,3′-triiodo-L-thyronine (T3), a hormone analogue derived by tissue deiodination of circulating L-thyroxine (T4). However, the ability of T4 and T3 to activate intracellular signal transduction cascades, independently of TR, has recently been described by several laboratories. Acting independently of TR, thyroid hormone also modulates activity of the plasma membrane Na+/H+ exchanger, Ca2+-stimulable ATPase, several other ion pumps or channels, and GTPase activity of synaptosomes. Studies from several laboratories have demonstrated the ability of thyroid hormone to activate the MAPK signal transduction cascade. These pathways typically are activated by physical and chemical signals at the cell surface. Although the kinetics and analog specificity for binding of thyroid hormone to the plasma membrane have been repeatedly reported, a cell surface receptor that accounts for these TR-dependent actions for thyroid hormone has not been previously identified.
Our laboratory has shown in the CV-1 monkey fibroblast cell line, which lacks functional TR, and in other cells that T4 activates the mitogen-activated protein kinase (MAPK; ERK112) signaling cascade and promotes the phosphorylation and nuclear translocation of MAPK as early as 10 min following application of a physiological concentration of T4. In nuclear fractions of thyroid hormone-treated cells, we have described complexes of activated MAPK and transactivator nucleoproteins that are substrates for the serine kinase activity of MAPK. These proteins include signal transducer and activator of transcription (STAT)-1α, STAT3, p53, estrogen receptor (ER)-α and, in cells containing TR, the nuclear thyroid hormone receptor for T3 (TRβ1). Thyroid hormone-directed MAPK-dependent phosphorylation of these proteins enhances their transcriptional capabilities. The effects of T4-induced MAPK activation are blocked by inhibitors of the MAPK signal transduction pathway and by tetraiodothyroacetic acid (tetrac), a thyroid hormone analog which inhibits Tq binding to the cell surface. Thyroid hormone-activated MAPK may also act locally at the plasma membrane, e.g., on the N+/H+ antiporter, rather than when translocated to the cell nucleus. A cell surface receptor for T4, that is linked to activation of the MAPK cascade has not previously been identified.
Integrins are a family of transmembrane glycoproteins that form noncovalent heterodimers. Extracellular domains of the integrins interact with a variety of ligands, including extracellular matrix glycoproteins, and the intracellular domain is linked to the cytoskeleton. Thyroid hormone was shown a decade ago to influence the interaction of integrin with the extracellular matrix protein, laminin, but the mechanism was not known. Integrin αVβ3 has a large number of extracellular protein ligands, including growth factors, and upon ligand-binding can activate the MAPK cascade. Several of the integrins contain an Arg-Gly-Asp (“RGD”) recognition site that is important to the liganding of matrix and other extracellular proteins that contain an Arg-Gly-Asp sequence.
Thus, it would be desirable to identify and provide an initiation site for the induction of MAPK signaling cascades in cells treated with thyroid hormones, or analogs and polymers thereof, thereby providing for methods of modulating growth factors and other polypeptides whose cell surface receptors clustered around this initiation site.
It is estimated that five million people are afflicted with chronic stable angina in the United States. Each year 200,000 people under the age of 65 die with what is termed “premature ischemic heart disease.” Despite medical therapy, many go on to suffer myocardial infarction and debilitating symptoms prompting the need for revascularization with either percutaneous transluminal coronary angioplasty or coronary artery bypass surgery. It has been postulated that one way of relieving myocardial ischemia would be to enhance coronary collateral circulation.
Correlations have now been made between the anatomic appearance of coronary collateral vessels (“collaterals”) visualized at the time of intracoronary thrombolitic therapy during the acute phase of myocardial infarction and the creatine kinase time-activity curve, infarct size, and aneurysm formation. These studies demonstrate a protective role of collaterals in hearts with coronary obstructive disease, showing smaller infarcts, less aneurysm formation, and improved ventricular function compared with patients in whom collaterals were not visualized. When the cardiac myocyte is rendered ischemic, collaterals develop actively by growth with DNA replication and mitosis of endothelial and smooth muscle cells. Once ischemia develops, these factors are activated and become available for receptor occupation, which may initiate angiogenesis after exposure to exogenous heparin. Unfortunately, the “natural” process by which angiogenesis occurs is inadequate to reverse the ischemia in almost all patients with coronary artery disease.
During ischemia, adenosine is released through the breakdown of ATP. Adenosine participates in many cardio-protective biological events. Adenosine has a role in hemodynamic changes such as bradycardia and vasodilation, and adenosine has been suggested to have a role in such unrelated phenomena as preconditioning and possibly the reduction in reperfusion injury (Ely and Beme, Circulation, 85: 893 (1992).
Angiogenesis is the development of new blood vessels from preexisting blood vessels (Mousa, S. A., In Angiogenesis Inhibitors and Stimulators: Potential Therapeutic Implications, Landes Bioscience, Georgetown, Tex.; Chapter 1, (2000)). Physiologically, angiogenesis ensures proper development of mature organisms, prepares the womb for egg implantation, and plays a key role in wound healing. The development of vascular networks during embryogenesis or normal and pathological angiogenesis depends on growth factors and cellular interactions with the extracellular matrix (Breier et al., Trends in Cell Biology 6:454-456 (1996); Folkman, Nature Medicine 1:27-31 (1995); Risau, Nature 386:671-674 (1997). Blood vessels arise during embryogenesis by two processes: vasculogenesis and angiogenesis (Blood et al., Bioch. Biophys. Acta 1032:89-118 (1990). Angiogenesis is a multi-step process controlled by the balance of pro- and anti-angiogenic factors. The latter stages of this process involve proliferation and the organization of endothelial cells (EC) into tube-like structures. Growth factors such as FGF2 and VEGF are thought to be key players in promoting endothelial cell growth and differentiation.
Control of angiogenesis is a complex process involving local release of vascular growth factors (P Carmeliet, Ann NY Acad Sci 902:249-260, 2000), extracellular matrix, adhesion molecules and metabolic factors (R J Tomanek, G C Schatteman, Anat Rec 261:126-135, 2000). Mechanical forces within blood vessels may also play a role (O Hudlicka, Molec Cell Biochem 147:57-68, 1995). The principal classes of endogenous growth factors implicated in new blood vessel growth are the fibroblast growth factor (FGF) family and vascular endothelial growth factor (VEGF) (G Pages, Ann NY Acad Sci 902:187-200, 2000). The mitogen-activated protein kinase (MAPK; ERK1/2) signal transduction cascade is involved both in VEGF gene expression and in control of proliferation of vascular endothelial cells.
Intrinsic adenosine may facilitate the coronary flow response to increased myocardial oxygen demands and so modulate the coronary flow reserve (Ethier et al., Am. J. Physiol., H131 (1993) demonstrated that the addition of physiological concentrations of adenosine to human umbilical vein endothelial cell cultures stimulates proliferation, possibly via a surface receptor. Adenosine may be a factor for human endothelial cell growth and possibly angiogenesis. Angiogenesis appears to be protective for patients with obstructive blood flow such as coronary artery disease (“CAD”), but the rate at which blood vessels grow naturally is inadequate to reverse the disease. Thus, strategies to enhance and accelerate the body's natural angiogenesis potential should be beneficial in patients with CAD.
Similarly, wound healing is a major problem in many developing countries and diabetics have impaired wound healing and chronic inflammatory disorders, with increased use of various cyclooxygenase-2 (CoX2) inhibitors. Angiogenesis is necessary for wound repair since the new vessels provide nutrients to support the active cells, promote granulation tissue formation and facilitate the clearance of debris. Approximately 60% of the granulation tissue mass is composed of blood vessels which also supply the necessary oxygen to stimulate repair and vessel growth. It is well documented that angiogenic factors are present in wound fluid and promote repair while antiangiogenic factors inhibit repair. Wound angiogenesis is a complex multi-step process. Despite a detailed knowledge about many angiogenic factors, little progress has been made in defining the source of these factors, the regulatory events involved in wound angiogenesis and in the clinical use of angiogenic stimulants to promote repair. Further complicating the understanding of wound angiogenesis and repair is the fact that the mechanisms and mediators involved in repair likely vary depending on the depth of the wound, type of wound (burn, trauma, etc.), and the location (muscle, skin, bone, etc.). The condition and age of the patient (diabetic, paraplegic, on steroid therapy, elderly vs infant, etc) can also determine the rate of repair and response to angiogenic factors. The sex of the patient and hormonal status (premenopausal, post menopausal, etc.) may also influence the repair mechanisms and responses. Impaired wound healing particularly affects the elderly and many of the 14 million diabetics in the United States. Because reduced angiogenesis is often a causative agent for wound healing problems in these patient populations, it is important to define the angiogenic factors important in wound repair and to develop clinical uses to prevent and/or correct impaired wound healing.
Thus, there remains a need for an effective therapy in the way of angiogenic agents as either primary or adjunctive therapy for promotion of wound healing, coronary angiogenesis, or other angiogenic-related disorders, with minimum side effects. Such a therapy would be particularly useful for patients who have vascular disorders such as myocardial infarctions, stroke or peripheral artery diseases and could be used prophylactically in patients who have poor coronary circulation, which places them at high risk of ischemia and myocardial infarctions.
Thyroid hormones, analogs, and polymeric conjugations play important roles in the development of the brain. Increasing evidence suggests that the deprivation of polymeric thyroid hormones in the early developmental stage causes structural and functional deficits in the CNS, but the precise mechanism underlying this remains elusive.
The mammalian nervous system comprises a peripheral nervous system (PNS) and a central nervous system (CNS, comprising the brain and spinal cord), and is composed of two principal classes of cells: neurons and glial cells. The glial cells fill the spaces between neurons, nourishing them and modulating their function. Certain glial cells, such as Schwann cells in the PNS and oligodendrocytes in the CNS, also provide a myelin sheath that surrounds neural processes. The myelin sheath enables rapid conduction along the neuron. In the peripheral nervous system, axons of multiple neurons may bundle together in order to form a nerve fiber. These, in turn, may be combined into fascicles or bundles.
During development, differentiating neurons from the central and peripheral nervous systems send out axons that grow and make contact with specific target cells. In some cases, axons must cover enormous distances; some grow into the periphery, whereas others are confined within the central nervous system. In mammals, this stage of neurogenesis is complete during the embryonic phase of life and neuronal cells do not multiply once they have fully differentiated.
A host of neuropathies have been identified that affect the nervous system. The neuropathies, which may affect neurons themselves or associated glial cells, may result from cellular metabolic dysfunction, infection, exposure to toxic agents, autoimmunity, malnutrition, or ischemia. In some cases, the cellular neuropathy is thought to induce cell death directly. In other cases, the neuropathy may induce sufficient tissue necrosis to stimulate the body's immune/inflammatory system and the immune response to the initial injury then destroys neural pathways.
Where the damaged neural pathway results from CNS axonal damage, autologous peripheral nerve grafts have been used to bridge lesions in the central nervous system and to allow axons to make it back to their normal target area. In contrast to CNS neurons, neurons of the peripheral nervous system can extend new peripheral processes in response to axonal damage. This regenerative property of peripheral nervous system axons is thought to be sufficient to allow grafting of these segments to CNS axons. Successful grafting appears to be limited, however, by a number of factors, including the length of the CNS axonal lesion to be bypassed, and the distance of the graft sites from the CNS neuronal cell bodies, with successful grafts occurring near the cell body.
Within the peripheral nervous system, this cellular regenerative property of neurons has limited ability to repair function to a damaged neural pathway. Specifically, the new axons extend randomly, and are often misdirected, making contact with inappropriate targets that can cause abnormal function. For example, if a motor nerve is damaged, regrowing axons may contact the wrong muscles, resulting in paralysis. In addition, where severed nerve processes result in a gap of longer than a few millimeters, e.g., greater than 10 millimeters (mm), appropriate nerve regeneration does not occur, either because the processes fail to grow the necessary distance, or because of misdirected axonal growth. Efforts to repair peripheral nerve damage by surgical means has met with mixed results, particularly where damage extends over a significant distance. In some cases, the suturing steps used to obtain proper alignment of severed nerve ends stimulates the formulation of scar tissue which is thought to inhibit axon regeneration. Even where scar tissue formation has been reduced, as with the use of nerve guidance channels or other tubular prostheses, successful regeneration generally still is limited to nerve damage of less than 10 millimeters in distance. In addition, the reparative ability of peripheral neurons is significantly inhibited where an injury or neuropathy affects the cell body itself or results in extensive degeneration of a distal axon.
Mammalian neural pathways also are at risk due to damage caused by neoplastic lesions. Neoplasias of both the neurons and glial cells have been identified. Transformed cells of neural origin generally lose their ability to behave as normal differentiated cells and can destroy neural pathways by loss of function. In addition, the proliferating tumors may induce lesions by distorting normal nerve tissue structure, inhibiting pathways by compressing nerves, inhibiting cerbrospinal fluid or blood supply flow, and/or by stimulating the body's immune response. Metastatic tumors, which are a significant cause of neoplastic lesions in the brain and spinal cord, also similarly may damage neural pathways and induce neuronal cell death.
One type of morphoregulatory molecule associated with neuronal cell growth, differentiation and development is the cell adhesion molecule (“CAM”), most notably the nerve cell adhesion molecule (N-CAM). The CAMs are members the immunoglobulin super-family. They mediate cell-cell interactions in developing and adult tissues through homophilic binding, i.e., CAM-CAM binding on apposing cells. A number of different CAMs have been identified. Of these, the most thoroughly studied are N-CAM and L-CAM (liver cell adhesion molecules), both of which have been identified on all cells at early stages of development, as well as in different adult tissues. In neural tissue development, N-CAM expression is believed to be important in tissue organization, neuronal migration, nerve-muscle tissue adhesion, retinal formation, synaptogenesis, and neural degeneration. Reduced N-CAM expression also is thought to be associated with nerve dysfunction. For example, expression of at least one form of N-CAM, N-CAM-180, is reduced in a mouse demyelinating mutant. Bhat, Brain Res. 452: 373-377 (1988). Reduced levels of N-CAM also have been associated with normal pressure hydrocephalus, Werdelin, Acta Neurol. Scand. 79: 177-181 (1989), and with type II schizophrenia. Lyons, et al., Biol. Psychiatry 23: 769-775 (1988). In addition, antibodies against N-CAM have been shown to disrupt functional recovery in injured nerves. Remsen, Exp. Neurobiol. 110: 268-273 (1990).
Currently no satisfactory method exists to repair the damage caused by traumatic injuries of motor neurons and diseases of motor neurons. There are 15,000 to 18,000 new cases of spinal cord injury each year in the United States. In addition, there are approximately 200,000 survivors of spinal cord injury. The annual cost of care for these patients exceeds $7 billion. The pathophysiology following acute spinal cord trauma is a complex and not fully understood mechanism. The primary tissue damage caused by mechanical trauma occurs immediately and is irreversible. Allen, J. Am. Med. Assoc. 57: 878-880 (1911). Experimental evidence indicates that much of the post-traumatic tissue damage is the result of a reactive process that begins within minutes after the injury and continues for days or weeks. Janssen, et al., Spine 14: 23-32 (1989) and Panter, et al., (1992). This progressive, self-destructive process includes pathophysiological mechanisms such as hemorrhage, post-traumatic ischemia, edema, axonal and neuronal necrosis, and demyelinization followed by cyst formation and infarction. For review, see Tator, et al., J. Neurosurg, 75: 15-26 (1991) and Faden, Crit. Rev. Neurobiol. 7: 175-186 (1993). Proposed injurious factors include electrolyte changes whereby increased intracellular calcium initiates a cascade of events (Young, J. Neurotrauma 9, Suppl. 1: S9-S25 (1992) and Young, J. Emerg. Med. 11: 13-22 (1993)), biochemical changes with uncontrolled transmitter release (Liu, et al., Cell 66: 807-815 (1991) and Yanase, et al., J. Neurosurg 83: 884-888 (1995), arachidonic acid release, free-radical production, lipid peroxidation (Braughler, et al., J. Neurotrauma 9, Suppl. 1: S1-S7 (1992), eicosanoid production (Demediuk, et al., J. Neurosci. Res. 20: 115-121 (1988), endogenous opioids (Faden, et al., Ann Neurol. 17: 386-390 (1985), metabolic changes including alterations in oxygen and glucose (Faden, Crit. Rev. Neurobiol. 7: 175-186 (1993)), inflammatory changes (Blight, J. Neurotrauma 9, Suppl. 1: S83-S91 (1992), and astrocytic edema (Kimelberg, J. Neurotrauma 9, Suppl. 1: S71-S81 (1992). For the past 400 years surgical approaches including laminectomy and decompression, accompanied by fusion, have been the most commonly practiced treatment strategies. Hansebout, “Early Management of Acute Spinal Cord Injury”, pp. 181-196 (1982) and Janssen, et al., Spine 14: 23-32 (1989). However, these procedures have not involved the application of techniques to augment the regenerative properties of spinal cord tissue.
A host of diseases of motor neurons have been identified, including demyelinating diseases, myelopathies, and diseases of motor neurons such as amyotrophic lateral sclerosis (“ALS”). INTERNAL MEDICINE, ch. 121-123 (4th ed., J. H. Stein, ed., Mosby, 1994). Multiple sclerosis (“MS”) is the most common demyelinating disorder of the central nervous system, causing patches of sclerosis (i.e., plaques) in the brain and spinal cord. MS has protean clinical manifestations, depending upon the location and size of the plaque. Typical symptoms include visual loss, diplopia, nystagmus, dysarthria, weakness, paresthesias, bladder abnormalities, and mood alterations. Myriad treatments have been proposed for this long-term variable illness. The list of proposed treatments encompasses everything from diet to electrical stimulation to acupuncture, emotional support, and various forms of immunosupressive therapy. None have proved to be satisfactory.
Progressive loss of lower and upper motor neurons occurs in several diseases (e.g., primary lateral sclerosis, spinal muscular atrophy, benign focal amyotrophy). However, ALS is the most common form of motor neuron disease. Loss of both lower and upper motor neurons occur in ALS. Symptoms include progressive skeletal muscle wasting, weakness, gasciculations, and cramping. Some cases have predominant involvement of brainstem motoneurons (progressive bulbar palsy). Unfortunately, treatment of motor neuron and related disease is largely supportive at this time. INTERNAL MEDICINE, ch. 123 (4th ed., J. H. Stein, ed., Mosby, 1994).
Accordingly, there is a need in the art for treatments of motor neurons disorders and injuries, and related deficits in neural functions. It is, therefore, an object of the present invention to provide compositions and methods for stimulating angiogenesis, for inducing neuronal differentiation, and for preventing the death or degeneration of neuronal cells.
The tyrosines are iodinated at one (monoiodotyrosine) or two (diiodotyrosine) sites and then coupled to form the active hormones (diiodotyrosine+diiodotyrosine→tetraiodothyronine [thyroxine, T4]; diiodotyrosine+monoiodotyrosine→triiodothyronine [T3]. Another source of T3 within the thyroid gland is the result of the outer ring deiodination of T4 by a selenoenzyme: type I 5′-deiodinase (5′D-I). Thyroglobulin, a glycoprotein containing T3 and T4 within its matrix, is taken up from the follicle as colloid droplets by the thyroid cells.
Lysosomes containing proteases cleave T3 and T4 from thyroglobulin, resulting in release of free T3 and T4. The iodotyrosines (monoiodotyrosine and diiodotyrosine) are also released from thyroglobulin, but only very small amounts reach the bloodstream. Iodine is removed from them by intracellular deiodinases, and this iodine is used by the thyroid gland.
The T4 and T3 released from the thyroid by proteolysis reach the bloodstream, where they are bound to thyroid hormone-binding serum proteins for transport. The major thyroid hormone-binding protein is thyroxine-binding globulin (“TBG”), which has high affinity but low capacity for T4 and T3. TBG normally accounts for about 75% of the bound hormones. Other thyroid hormone-binding proteins—primarily thyroxine-binding prealbumin, also called transthyretin (“TTR”), which has high affinity but low capacity for T4, and albumin, which has low affinity but high capacity for T4 and T3-account for the remainder of the bound serum thyroid hormones. About 0.03% of the total serum T4 and 0.3% of the total serum T3 are free and in equilibrium with the bound hormones. Only free T4 and T3 are available to the peripheral tissues for thyroid hormone action.
Thyroid hormones have two major physiologic effects: (1) They increase protein synthesis in virtually every body tissue. (T3 and T4 enter cells, where T3, which is derived from the circulation and from conversion of T4 to T3 within the cell, binds to discrete nuclear receptors and influences the formation of mRNA.) (2) T3 increases O2 consumption by increasing the activity of the Na+, K+-ATPase (Na pump), primarily in tissues responsible for basal O2 consumption (ie, liver, kidney, heart, and skeletal muscle). The increased activity of Na+, K+-ATPase is secondary to increased synthesis of this enzyme; therefore, the increased O2 consumption is also probably related to the nuclear binding of thyroid hormones. However, a direct effect of T3 on the mitochondrion has not been ruled out. T3 is believed to be the active thyroid hormone, although T4 itself may be biologically active.
The pool of thyroid hormones critical for the biological actions of the hormones is the pool of free thyroid hormone. The size of this pool is determined for short time periods by uptake/release of thyroid hormones into/from cell and binding/release of thyroid hormones by thyroid hormone-binding proteins. Both proportions and absolute concentrations of these proteins differ in blood plasma and cerebrospinal fluid (“CSF”). The most pronounced difference is found for transthyretin (“TTR”), which is the only thyroid hormone-binding plasma protein synthesized in the brain (Schreiber G, Southwell B R, Richardson S J. Hormone delivery systems to the brain-transthyretin. Exp Clin Endocrinol Diabetes. 1995; 103(2):75-80). TTR is also distinct from the other two thyroid hormone-binding plasma proteins in humans by the absence of genetic deficiencies. TTR gene expression was initiated during evolution much earlier in the brain than in the liver. The structure of the domains of TTR involved in thyroxine (TR) T4 binding has been completely conserved for 350 million years. These observations point to a special functional significance of TTR in the brain. It is proposed that this is the determination of the level of free T4 in the extracellular compartment of the brain. T4 can then be converted in the brain to triiodothyronine T3 by specific deiodinases. This T3 can interact with receptors in the cell nuclei, regulating gene transcription.
Alzheimer's disease is a severe neurodegenerative disorder, and currently about 4 million Americans suffer from this disease. As the aging population continues to grow, this number could reach 14 million by the middle of next century unless a cure or prevention is found. At present, there is no sensitive and specific premortem test for early diagnosis of this disease. Alzheimer's disease is currently diagnosed based on the clinical observation of cognitive decline, coupled with the systematic elimination of other possible causes of those symptoms. The confirmation of the clinical diagnosis of “probable Alzheimer's disease” can only be made by examination of the postmortem brain. The Alzheimer's disease brain is characterized by the appearance of two distinct abnormal proteinaceous deposits in regions of the brain responsible for learning and memory (e.g., cerebral cortex and hippocampus). These deposits are extracellular amyloid plaques, which are characteristic of Alzheimer's disease, and intracellular neurofibillary tangles (“NFTs”), which can be found in other neurodegenerative disorders as well. Amyloid peptides are typically either 40 or 42 amino acids in length (“A1-40” or “A1-42”, respectively) and are formed from abnormal processing of a larger membrane-associated protein of unknown function, the amyloid precurser protein (“APP”). Oligomeric aggregates of these peptides are thought to be neurotoxic, eventually resulting in synaptic degeneration and neuronal loss. The amount of amyloid deposition roughly correlates with the severity of symptoms at the time of death.
In the past, there have been several attempts for the design of radiopharmaceuticals that could be used as diagnostic agents for a premortem diagnosis of Alzheimer's disease. Bomebroek et al. showed that the amyloid-associated protein serum amyloid P component (SAP), labeled with 123I, accumulates at low levels in the cerebral cortex, possibly in vessel walls, of patients with cerebral amyloidosis (Bomebroek, M., et al., Nucl. Med. Commun. (1996), Vol. 17, pp. 929-933).
Saito et al. proposed a vector-mediated delivery of 123I-labeled A1-40 through the blood-brain barrier. It is reported that the iodinated A1-40 binds A amyloid plaque in tissue sections (Saito, Y., et al., Proc. Natl. Acad. Sci. USA 1995, Vol. 92, pp. 10227-10231). U.S. Pat. No. 5,231,000 discloses antibodies with specificity to A4 amyloid polypeptide found in the brain of Alzheimer's disease patients. However, a method to deliver these antibodies across the blood-brain barrier has not been described. Zhen et al. described modifications of the amyloid-binding dye known as “Congo Red™”, and complexes of these modified molecules with technetium and rhenium. The complexes with radioactive ions are purported to be potential imaging agents for Alzheimer's disease (Zhen et al., J. Med. Chem. (1999), Vol. 42, pp. 2805-2815). However, the potential of the complexes to cross the blood-brain barrier is limited.
A group at the University of Pennsylvania in the U.S.A. (Skovronsky, M., et al., Proc. Natl. Acad. Sci. 2000, Vol. 97, pp. 7609-7614) has developed a fluorescently labeled derivative of Congo Red that is brain permeable and that non-specifically binds to amyloid materials (that is, peptides in-pleated sheet conformation). This compound would need to be radiolabeled and then run through pre-clinical screens for pharmacokinetics and toxicity before clinical testing. In contrast, our invention utilizes derivatives of naturally occurring substances alone or in combinations for the diagnosis, prevention, and treatment of Alzheimer's disease. Klunk et al. reported experiments with a derivative of Congo Red™, Chrysamine G (“CG”). It is reported that CG binds synthetic-amyloid well in vitro, and crosses the blood-brain barrier in normal mice (Klunk et al., Neurobiol. Aging (1994), Vol. 15, No. 6, pp. 691-698). Bergstrom et al. presented a compound labeled with 123I as a potential radioligand for visualization of M1 and M2 muscarinic acetylcholine receptors in Alzheimer's disease (Bergstrom et al., Eur. J. Nucl. Med. (1999), Vol. 26, pp. 1482-1485).
Recently, it has been discovered that certain specific chemokine receptors are upregulated in the brains of patients with Alzheimer's disease (Horuk, R. et al., J. Immunol. (1997), Vol. 158, pp. 2882-2890); Xia et al., J. Neuro Virol (1999), Vol. 5, pp. 32-41). In addition, it has been recently shown that the chemokine receptor CCR1 is upregulated in the brains of patients with advanced Alzheimer's disease and absent in normal-aged brains (Halks-Miller et al, CCR1 Immunoreactivity in Alzheimer's Disease Brains, Society for Neuroscience Meeting Abstract, #787.6, Volume 24, 1998). Antagonists to the CCR1 receptor and their use as anti-inflammatory agents are described in the PCT Published Patent Application, WO 98/56771.
None of the above described proposals have resulted in a clinical development of an imaging agent for the early diagnosis of Alzheimer's disease. Accordingly, there is still a clinical need for a diagnostic agent that could be used for a reliable and early diagnosis of Alzheimer's disease. Additionally, the proposed strategies would also be useful for the inhibition of amyloid plaque formation or buildup in Alzheimer patients. Accordingly, it is an object of the present invention to provide compositions and methods for the early diagnosis, prevention, and treatment of neurodegenerative diseases, such as, for example Alzheimer's disease.
It is interesting to note that angiogenesis also occurs in other situations, but which are undesirable, including solid tumour growth and metastasis; rheumatoid arthritis; psoriasis; scleroderma; and three common causes of blindness—diabetic retinopathy, retrolental fibroplasia and neovascular glaucoma (in fact, diseases of the eye are almost always accompanied by vascularization. The process of wound angiogenesis actually has many features in common with tumour angiogenesis. Thus, there are some conditions, such as diabetic retinopathy or the occurrence of primary or metastatic tumors, where angiogenesis is undesirable. Thus, there remains a need for methods by which to inhibit the effect of angiogenic agents for the treatment of cancers.